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Rat Epidermal Keratinocytes (REK) Culture Protocol

I. Storage

A. Cryopreserved Vials (R102-05)

Store the cryovials in a liquid nitrogen storage tank immediately upon arrival.

*Be sure to wear face protection mask and gloves when retrieving cryovials from the liquid nitrogen storage tank. The dramatic temperature change from the tank to the room could cause any trapped liquid nitrogen in the cryovials to burst and cause injury.

r102-05-cells

Rat Epidermal Keratinocytes (REK)

II. Preparation for Culturing

  1. Ensure the Class II Biological Safety Cabinet, with HEPA filtered laminar airflow, is in proper working condition.
  2. Sterilize the Biological Safety Cabinet with 70% alcohol.
  3. Turn the Biological Safety Cabinet blower on for 10 minutes before beginning cell culture work.
  4. Make sure all serological pipettes, pipette tips and reagent solutions are sterile.
  5. Follow the standard sterilization technique and safety rules:
    a. Do not pipette by mouth.
    b. Always wear gloves and safety glasses when working with animal cells even though all the rodents were
        provided and inspected by Harlan Sprague Dawley, Inc.
    c. Handle all cell culture work in a sterile hood.

III. Culturing REK

A. Preparing Cell Culture Flasks for Culturing REK

  1. Take the Rat Keratinocyte Growth Medium (R131-250) from the refrigerator. Decontaminate the bottle with 70% alcohol in a sterile hood.
  2. Pipette 15 ml of Rat Keratinocyte Growth Medium (R131-250)* to a T-75 flask (SIAL0641).
    * Keep the medium to surface area ratio at 1ml per 5 cm2.
    For example:
    -  5 ml for a T-25 flask (SIAL0639) or a 60 mm tissue culture dish (SIAL0166).
    -  15 ml for a T-75 flask (SIAL0641) or a 100 mm tissue culture dish (SIAL0167).

B. Thawing and Plating REK

  1. Remove the cryopreserved vial of REK from the liquid nitrogen storage tank using proper protection for your eyes and hands.
  2. Turn the vial cap a quarter turn to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
  3. Thaw the cells quickly by placing the lower half of the vial in a 37°C water bath and watch the vial closely during the thawing process.
  4. Remove the vial from the water bath when only a small amount of ice is left in the vial. Do not let cells thaw completely.
  5. Decontaminate the vial exterior with 70% alcohol in a sterile Biological Safety Cabinet.
  6. Remove the vial cap carefully. Do not touch the rim of the cap or the vial with your hands to avoid contamination.
  7. Resuspend the cells in the vial by gently pipetting the cells 5 times with a 2 ml pipette. Be careful not to pipette too vigorously as to cause foaming.
  8. Pipette the cell suspension (1ml) from the vial into the T-75 flask (SIAL0641) containing 15 ml of Rat Keratinocyte Growth Medium (R131-250).
  9. Cap the flask and rock gently to evenly distribute the cells.
  10. Place the T-75 flask (SIAL0641) in a 37oC, 5% CO2 humidified incubator. Loosen the cap to allow gas exchange. For best results, do not disturb the culture for 24 hours after inoculation.
  11. Change to fresh Rat Keratinocyte Growth Medium (R131-250) after 24 hours or overnight to remove all traces of DMSO.
  12. Change Rat Keratinocyte Growth Medium (R131-250) every other day until the cells reach 45% confluency.
  13. Double the Rat Keratinocyte Growth Medium (R131-250) volume when the culture is >45% confluent or for weekend feedings.

Materials

     
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